Chemokines are a family of related soluble proteins of molecular weight between 8 and 10 KDa, secreted by lymphocytes and other cells, which bind receptors on target cell surfaces resulting in the activation and mobilization of leukocytes, for example in the inflammatory process. Recently, Cocchi et al. demonstrated that the chemokines RANTES, MIP-1α and MIP-1β are factors produced by CD8+ T lymphocytes which inhibit infection by macrophage-tropic primary isolates of HIV-1, but not infection by laboratory-adapted strains of the virus (1). These chemokines are members of the C-C group of chemokines, so named because they have adjacent cysteine residues, unlike the C-X-C group which has a single amino acid separating these residues (2). While Cocchi et al. found that expression of HIV-1 RNA was suppressed by treatment with the chemokines, they did not identify the site of action of these molecules.
A resonance energy transfer (RET) assay of HIV-1 envelope glycoprotein-mediated membrane fusion was used to determine whether fusion mediated by the envelope glycoprotein from the primary macrophage-tropic isolate of HIV-1JR-FL would be specifically inhibited by chemokines, when compared with fusion mediated by the envelope glycoprotein from the laboratory-adapted T lymphotropic strain HIV-1LAI. As described below, it was demonstrated that this is indeed the case. This demonstrates that some chemokine receptors are fusion accessory molecules required for HIV-1 infection. Previous studies have indicated that unidentified cell surface molecules are required for virus entry in addition to the HIV-1 receptor, CD4. While CD4 is required for HIV-1 attachment, the accessory molecules are required for the membrane fusion step of entry. These accessory molecules are generally expressed only on human cells, so HIV-1 does not infect non-human CD4+ cells (3-6). Moreover it is possible to complement non-human CD4+ cells by fusing them (using polyethylene glycol) with CD4− human cells, resulting in a heterokaryon which is a competent target for HIV-1 envelope-mediated membrane fusion (7, 8). These studies have been performed using laboratory-adapted T lymphotropic strains of the virus.
In some cases, it appears that fusion accessory molecules are found on a subset of human CD4+ cells and are required for infection by HIV-1 isolates with particular tropisms. For example, macrophage-tropic primary strains of HIV-1 such as HIV-1JR-FL may have different requirements for accessory molecules compared with laboratory-adapted T lymphotropic strains such as HIV-1LAI. This phenomenon may explain differences in tropism between HIV-1 strains.
The current invention comprises a series of new therapeutics for HIV-1 infection. It was demonstrated for the first time that chemokines act at the fusion step of HIV-1 entry and specifically inhibit membrane fusion mediated by the envelope glycoprotein of primary macrophage-tropic primary viral isolates, not laboratory-adapted T lymphotrophic strains of the virus. Primary macrophage-tropic isolates of the virus are of particular importance since they are the strains usually involved in virus transmission, and may have particular importance in the pathogenesis of HIV-1 infection.
These results were obtained using a resonance energy transfer (RET) assay of HIV-1 envelope-mediated membrane fusion. Moreover, this assay is used to identify non-chemokines, including fragments of chemokines and modified chemokines, that inhibit HIV-1 envelope glycoprotein-mediated membrane fusion and thereby neutralize the virus, yet do not induce an inflammatory response.